The Asklev paleosol is a well developed Podzol in sandy till of late Saalian age ‐ the Asklev Till. The Asklev podsol is covered by fluvial sand in which another weaker podsol is present. Thermo-luminescense dating of the sand layer revealed an age of c. 100 ka BP, i.e. that the soil surface was buried in the early Weichelian. The surface was thus stable during the entire Eemain interglacial and subject to pedogenesis for >15.000 years. Discordantly resting on the fluvial sand is about 1.5 m of sandy till with an undisturbed grey lower part and a brown cryoturbated upper part. Fabric analyses from the lower grey part of the till reveal an ice movement from the SSE. This till is deposited during MIS 4 in middle Weichselian Thin sections from the Podzol’ show that the buried A-horizons micromorphology is not fully comparable to present-day Podzols in the region as it has a well developed argillic horizon below despite the parent material low in clay (< 3%). In contrast to modern Danish Podzols it also retain ample evidence of burning (charcoal) and frost features (capping). The Asklev paleosol classify as a Placic Podzol, but is a typical bi-sequum with a Bt-horizon at depth. Its content of organic C is up to 38 mg C/g soil in the A-horizon, 8.4 mg C/g soil in the Bhs-horizon, which decreases to <1 mg C/g soil in the C-horizon. Carbon-to-nitrogen ratios range from 80 in the E-horizon to 25 in the C-horizon. Concentrations of heavy metals is low with maximums of 3.7 mg Ni/kg soil, 16.4 mg Cr/kg soil, <0.05 mg Cd/kg soil, 6.7 mg Pb/kg soil and 33 mg Cu/kg soil. Arsenic concentrations vary from <0.1 mg/kg soil in the E-horizon to 4.7 mg/kg soil in the Bt-horizon. Comparing the geochemistry of the Asklev paleosol with a modern analogue Podzol in a nearby natural, ancient woodland reveal a general higher content of heavy metals in the paleosol. These differences reflect a combination of different parent materials, modern-day pollution rates, losses/additions during the c. 90 ka where the soil has experienced permafrost, and the c. 10 ka with moist conditions during the present interglacial. The combined methods together reflect a soil surface from the previous interglacial which may act as an important reference for modern-day soil chemical status and e.g. pollution rates, especially if enough sites with the Asklev paleosol is analysed.

The Asklev paleosol is a well developed Podzol in sandy till of late Saalian age - the Asklev Till. The Asklev podsol is covered by fluvial sand in which another weaker podsol is present. Thermo-luminescense dating of the sand layer revealed an age of c. 100 ka BP, i.e. that the soil surface was buried in the early Weichelian. The surface was thus stable during the entire Eemain interglacial and subject to pedogenesis for >15.000 years. Discordantly resting on the fluvial sand is about 1.5 m of sandy till with an undisturbed grey lower part and a brown cryoturbated upper part. Fabric analyses from the lower grey part of the till reveal an ice movement from the SSE. This till is deposited during MIS 4 in middle Weichselian Thin sections from the Podzol' show that the buried A-horizons micromorphology is not fully comparable to present-day Podzols in the region as it has a well developed argillic horizon below despite the parent material low in clay (< 3%). In contrast to modern Danish Podzols it also retain ample evidence of burning (charcoal) and frost features (capping). The Asklev paleosol classify as a Placic Podzol, but is a typical bi-sequum with a Bt-horizon at depth. Its content of organic C is up to 38 mg C/g soil in the A-horizon, 8.4 mg C/g soil in the Bhs-horizon, which decreases to <1 mg C/g soil in the C-horizon. Carbon-to-nitrogen ratios range from 80 in the E-horizon to 25 in the C-horizon. Concentrations of heavy metals is low with maximums of 3.7 mg Ni/kg soil, 16.4 mg Cr/kg soil, <0.05 mg Cd/kg soil, 6.7 mg Pb/kg soil and 33 mg Cu/kg soil. Arsenic concentrations vary from <0.1 mg/kg soil in the E-horizon to 4.7 mg/kg soil in the Bt-horizon. Comparing the geochemistry of the Asklev paleosol with a modern analogue Podzol in a nearby natural, ancient woodland reveal a general higher content of heavy metals in the paleosol. These differences re- flect a combination of different parent materials, modern-day pollution rates, losses/additions during the c. 90 ka where the soil has experienced permafrost, and the c. 10 ka with moist conditions during the present interglacial. The combined methods together reflect a soil surface from the previous interglacial which may act as an important reference for modern-day soil chemical status and e.g. pollution rates, especially if enough sites with the Asklev paleosol is analysed.

An aqueous extract of the saponin drugQuillaja saponaria Molina was separated by a combination of gel exclusion and ion exchange chromatography. Some of the fractions were investigated for adjuvant activity in guinea pigs. The guinea pigs were immunized against foot-and-mouth disease virus serotype 0, and adjuvant activity was estimated as an increase in serum neutralizing antibodies and in resistance to challenge with homologous virus. Only one fraction showed adjuvant activity and this was confirmed in cattle. The animals were immunized with a stock vaccine foot-and-mouth disease serotype 0 of the Frenkel type and adjuvant activity was estimated as an increase in serum neutralizing antibodies. The fraction was subjected to thin-layer chromatography, polyacrylamide gel electrophoresis, and analytical ultracentrifugation. These analytical techniques indicated that only one substance was present in the fraction. It was shown that this substance in aqueous solutions was present in a micellar state above a certain critical micelle concentration.

Anthropogenic sandy soils in Scotland, Denmark and The Netherlands were studied to determine the extent to which they had been influenced by inputs of materials and current land management. Particle size distribution, pH, loss on ignition, ECEC, base saturation and total P were determined for eight profiles under different land uses (arable, meadow and woodland). Inputs of materials were identified by comparison of particle size distributions of the deepened cultivated horizons with those of parent materials. At all three sites turf derived from local sources was the main input though there were other mineral additions including an aeolian component. Overall, the anthropogenic soils are remarkably similar in field and some analytical properties, though land use has influenced pH, base saturation and total P.

Dating of prehistoric anthropogenic earthworks requires either excavation for archaeological artifacts or macroscopic organic matter suitable for 14C analysis. Yet, the former, in many cases, is undesirable and the latter is difficult to obtain. Here we present a soil science procedure, which has the potential to overcome these problems. It includes careful sampling of buried former soil surfaces, acid-alkali-acid fractionation of soil organic matter (SOM), and subsequent 14C AMS dating. To test the procedure, soil from one of the largest known burial mounds in Scandinavia, Hohøj, and 9 other Danish burial mounds were sampled. The 14C dates from extracted SOM fractions were compared to reference ages obtained by other methods. We show that humic acid fractions in 7 of the 10 mounds had the same age as the reference, or were, at maximum, 280 yr older than the reference ages. The best age estimates were derived from an organic-rich layer from the upper cm of buried soil or sod. Differences among SOM fraction ages probably indicate the reliability of the dating. Hohøj dated to approximately 1400 BC and, thus, was up to 500 yr older than other dated Scandinavian mounds of comparable size. The remaining investigated burial mounds were dated to between 1700 and 1250 BC. We conclude that combined sampling of buried soil surfaces, SOM fractionation, and 14C analysis allows for dating of archaeological earthworks when minimal disturbance is required, or if no macroscopic organic remains are found.

Mucosally active vaccine adjuvants that will prime a full range of local and systemic immune responses against defined antigenic epitopes are much needed. Cholera toxin and lipophilic immune stimulating complexes (ISCOMS) containing Quil A can both act as adjuvants for orally administered Ags, possibly by targeting different APCs. Recently, we have been successful in separating the adjuvant and toxic effects of cholera toxin by constructing a gene fusion protein, CTA1-DD, that combines the enzymatically active CTA1-subunit with a B cell-targeting moiety, D, derived from Staphylococcus aureus protein A. Here we have extended this work by combining CTA1-DD with ISCOMS, which normally target dendritic cells and/or macrophages. ISCOMS containing a fusion protein comprising the OVA323–339 peptide epitope linked to CTA1-DD were highly immunogenic when given in nanogram doses by the s.c., oral, or nasal routes, inducing a wide range of T cell-dependent immune responses. In contrast, ISCOMS containing the enzymatically inactive CTA1-R7K-DD mutant protein were much less effective, indicating that at least part of the activity of the combined vector requires the ADP-ribosylating property of CTA1. No toxicity was observed by any route. To our knowledge, this is the first report on the successful combination of two mechanistically different principles of adjuvant action. We conclude that rationally designed vectors consisting of CTA1-DD and ISCOMS may provide a novel strategy for the generation of potent and safe mucosal vaccines.

A vaccine based upon a recombinant plant virus (CPMV-PARVO1), displaying a peptide derived from the VP2 capsid protein of canine parvovirus (CPV), has previously been described. To date, studies with the vaccine have utilized viable plant chimaeric particles (CVPs). In this study, CPMV-PARVO1 was inactivated by UV treatment to remove the possibility of replication of the recombinant plant virus in a plant host after manufacture of the vaccine. We show that the inactivated CVP is able to protect dogs from a lethal challenge with CPV following parenteral immunization with the vaccine. Dogs immunized with the inactivated CPMV-PARVO1 in adjuvant displayed no clinical signs of disease and shedding of CPV in faeces was limited following CPV challenge. All immunized dogs elicited high titres of peptide-specific antibody, which neutralized CPV in vitro. Levels of protection, virus shedding and VP2-specific antibody were comparable to those seen in dogs immunized with the same VP2- peptide coupled to keyhole limpet hemocyanin (KLH). Since plant virus-derived vaccines have the potential for cost-effective manufacture and are not known to replicate in mammalian cells, they represent a viable alternative to current replicating vaccine vectors for development of both human and veterinary vaccines.

The Ulfborg Project is investigating the interactions of natural and anthropogenic influences on a typical West Jutland landscape through the Holocene, with emphasis on the last millennium. Pollen analysis and AMS 14C dates of buried podzols were used to date periods of blown sand transport and the development of man-made Calluna vulgaris heath. Hydrochloric acid-extractable carbon, expected to represent the most mobile fraction of carbon derived from the youngest soil, was a non-negligible fraction of the total carbon in the buried soils. The age of the humic acid fraction in the Bs horizons seems to indicate the start of podzolisation, whereas the fulvic acid fraction in the mor layer indicates the time of burial. Dates for the three alkali-extracted fractions within the individual mor layers were similar. They agree with the results of pollen analyses, which indicate that the original forest was transformed into pure Calluna heath and kept as such by burning and cutting, thus preventing continuous accumulation of organic matter. The first large-scale sand movement event was dated to about 6000 BC, covering soils on which forest had earlier established. The vegetational disturbance and sand movement were probably triggered by a climatic change to cooler conditions. In contrast, later sand movement seems to be connected with human activity. In soils covered by sand at 800–600 BC the pollen record shows that the change from forest to pure Calluna vulgaris heath had already taken place. Together with evidence from lacustrine pollen records, the pollen assemblages from all younger mor layers indicate that the area was kept as pure Calluna heath until the 19th century. 14C dates of the lower Bs horizons indicate that podzolisation started soon after the sand was deposited, so some parent materials had been leached before deposition.

Mucosally active vaccine adjuvants that will prime a full range of local and systemic immune responses against defined antigenic epitopes are much needed. Cholera toxin and lipophilic immune stimulating complexes (ISCOMS) containing Quil A can both act as adjuvants for orally administered Ags, possibly by targeting different APCs. Recently, we have been successful in separating the adjuvant and toxic effects of cholera toxin by constructing a gene fusion protein, CTA1-DD, that combines the enzymatically active CTA1-subunit with a B cell-targeting moiety, D, derived from Staphylococcus aureus protein A. Here we have extended this work by combining CTA1-DD with ISCOMS, which normally target dendritic cells and/or macrophages. ISCOMS containing a fusion protein comprising the OVA(323-339) peptide epitope linked to CTA1-DD were highly immunogenic when given in nanogram doses by the s.c., oral, or nasal routes, inducing a wide range of T cell-dependent immune responses. In contrast, ISCOMS containing the enzymatically inactive CTA1-R7K-DD mutant protein were much less effective, indicating that at least part of the activity of the combined vector requires the ADP-ribosylating property of CTA1. No toxicity was observed by any route. To our knowledge, this is the first report on the successful combination of two mechanistically different principles of adjuvant action. We conclude that rationally designed vectors consisting of CTA1-DD and ISCOMS may provide a novel strategy for the generation of potent and safe mucosal vaccines.

Semipurified Quil A and purified Quil A were used to prepare well-characterized subunit vaccine candidates against measles. Variation in the relative amounts of the measles virus (MV) fusion (F) protein, Quil A-components and lipids did not influence induction of antibody responses in mice, but had a pronounced effect on the capacity to induce cytotoxic T cell (CTL) activity of a CD8+ MV F-protein specific human T cell clone in vitro. A characteristic MV iscom preparation based on the combined use of HPLC-purified Quil A-components QA-3 and QA-22 (QA-3/22) efficiently induced CTL activity in vitro. Comparable results were obtained by mixing β-propiolactone inactivated MV with iscom-matrix QA-3/22 or free QA-22. On the basis of the data presented it was concluded that these three preparations are interesting MV vaccine candidates for further evaluation in pre-clinical experiments in a primate model.

Immunisation against pathogens remains one of the most effective ways of preventing or reducing losses due to infectious diseases in animal husbandry. When inactivated vaccines are used, adjuvants are most often required to obtain satisfactory immune responses. One such type of adjuvant is saponin derived from the bark of Quillaja saponaria Molina, a tree of the rose family. A few different commercial sources exist, but due to the structural complexity and heterogeneity of these saponin preparations, it has been difficult to establish exactly which components are responsible for the adjuvant activity. By carefully selecting the bark source, we have succeeded in preparing a much less heterogeneous preparation of quillaja saponin. In this report we describe the preparation, in terms of structural complexity, hemolytic activity, adjuvant activity, and its ability to form ISCOM matrix. This new preparation could have implications for use per se, or as starting material for more effective preparation of pure substances.

Feline immunodeficiency virus (FIV) is a natural lentiviral pathogen of cats which can be experimentally transmitted via rectal and vaginal routes--the major routes of human immunodeficiency virus type 1 transmission in man. An important objective for lentiviral research is the development of vaccine strategies which generate good mucosal immune responses capable of giving protection from a mucosal virus challenge. The experimental vaccines employed in this study were based on (a) a peptide from the third variable region of the FIV envelope glycoprotein and (b) fixed whole FIV, Glasgow-8 strain. Adjuvants used were Quil A and cholera toxin for mucosal administration and incomplete Freund's adjuvant and immune stimulating complexes for subcutaneous injection. Mucosal immunization was given by rectal and intranasal routes. Both antibody and proliferative responses were elicited by mucosal immunization and cholera toxin was found to be a good mucosal adjuvant. The addition of a lipo thioester to the FIV peptide improved IgG and IgA responses upon parenteral administration. However, no protection from a rectal FIV challenge was achieved.

Bovine Viral Diarrhea Virus (BVDV) is a major pathogen of cattle in most countries. The main reservoir of virus in herds are BVDV persistently infected animals, which arise as a result of infection of the bovine fetus early in gestation. The spread of virus to the unborn fetus may be prevented by vaccination of the dam. We describe in this report the production and initial testing of an inactivated subunit vaccine against BVDV. The vaccine is based on production of antigen in primary bovine cell cultures, extraction of antigens from infected cells with detergent, chromatographic purification, concentration, and insertion of antigens into immune stimulating complexes (ISCOMs). Vaccines based on two different Danish strains of BVDV were injected into calves and the antisera produced were tested for neutralising activity against a panel of Danish BVDV strains. The two vaccines induced different neutralisation responses, which seem to partly complement each other. The implication of these observations for successful vaccination against BVDV is discussed.

African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned inEscherichia coliusing the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151–200) and II (residues 83–120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule. Neutralizing epitopes were defined at positions 85–92 (PDPLSPGE) for MAb 10AE12 and at 179–185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques. These data will be especially useful for vaccine development and diagnostic purposes.

ABSTRACT The humoral immune responses to the D2 peptide of fibronectin-binding protein B (FnBP) of Staphylococcus aureus , expressed on the plant virus cowpea mosaic virus (CPMV), were evaluated after mucosal delivery to mice. Intranasal immunization of these chimeric virus particles (CVPs), either alone or in the presence of ISCOM matrix, primed CPMV-specific T cells and generated high titers of CPMV- and FnBP-specific immunoglobulin G (IgG) in sera. Furthermore, CPMV- and FnBP-specific IgA and IgG could also be detected in the bronchial, intestinal, and vaginal lavage fluids, highlighting the ability of CVPs to generate antibody at distant mucosal sites. IgG2a and IgG2b were the dominant IgG subclasses in sera to both CPMV and FnBP, demonstrating a bias in the response toward the T helper 1 type. The sera completely inhibited the binding of human fibronectin to the S. aureus FnBP. Oral immunization of the CVPs also generated CPMV- and FnBP-specific serum IgG; however, these titers were significantly lower and more variable than those generated by the intranasal route, and FnBP-specific intestinal IgA was undetectable. Neither the ISCOM matrix nor cholera toxin enhanced these responses. These studies demonstrate for the first time that recombinant plant viruses have potential as mucosal vaccines without the requirement for adjuvant and that the nasal route is most effective for the delivery of these nonreplicating particles.

The antigenic structure of the capsid proteins of porcine parvovirus (PPV) was investigated. A total of nine linear epitopes were identified by Pepscan using porcine or rabbit anti-PPV antisera. No sites were identified with a panel of neutralising monoclonal antibodies (MAbs). All epitopes were located in the region corresponding to the major capsid protein VP2. Based on this information, and on analogy to other autonomous parvoviruses, 24 different peptides were synthesised, coupled to keyhole limpet haemocyanin (KLH) and used to immunise rabbits. Most antisera were able to bind viral protein. Only peptides from the N-terminal part of VP2 were able to induce virus-neutralising antibodies, although at low levels. A similar neutralising activity could be obtained in pigs. The exposure of the N-terminus was shown in full virions, both by immunoelectron microscopy and absorption experiments. It is concluded that in PPV, the VP2 N-terminus is involved in virus neutralisation (VN) and peptides from this region are therefore primary targets for developing peptide-based vaccines against this virus.

Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal antibodies (MAbs) and 12 overlapping fragments of the protein expressed in E, coli, Two antigenic regions were found. Ten out of the 11 MAbs recognized different discontinuous epitopes in the most immunodominant region of the viral capsid, This domain was located between residues 31 and 250 of the VP60 N terminus. The other MAb revealed the presence of an antigenic site within 102 aa of the C terminus. This MAb did not recognize the major cleavage product of the full-length 60 kDa protein. These results indicate that, in contrast to other caliciviruses such as Norwalk virus (NV), the 36 kDa cleavage product probably forms the N-terminal region of VP60. However, as in NV, the cleavage region appears to be the most immunodominant region.

Synthetic peptides have frequently been used to immunize animals. However, peptides less than about 20 to 30 amino acids long are poor immunogens. In general, to increase its immunogenicity, the presentation of the peptide should be improved, and molecular weight needs to be increased. Many attempts have been made to couple peptide immunogens to different carrier proteins [e.g. keyhole limpet haemocyanin (KLH) or ovalbumin]. This leads to very complex structures, however. We used a controlled conjugation of a peptide to a single long-chain fatty acid like palmitic acid by a thioester or an amide bond. It was found that these S-palmitoylated peptides were much more immunogenic than N-palmitoylated peptides and at least similar to KLH-conjugated peptides with respect to appearance and magnitude of induced antibodies (canine parvovirus) or immunocastration effect (gonadotropin-releasing hormone). For chemical synthesis of thioesters, we established conditions for solution and solid-phase synthesis. In both phases, Cys(SBut) could only be deprotected efficiently using phosphines, and S-acylation was accomplished using standard coupling at pH 5. We speculate that, in vivo, the presence of an appropriate fatty acid chain, chemically linked through a labile thioester bond, greatly enhances immunogenicity, because it represents a favourable substrate for cleavage by cellular thioesterases in cells of the immune system.